Micropropagation and field evaluation of seven strawberry genotypes suitable for agro-climatic condition of Bangladesh

Strawberry (Fragaria × ananassa Duch.) is one of the important and popular fruits in the temperate countries of the world due to its fragrance, taste and nutritional properties. Due to its popularity and increasing demand in Bangladesh, an experiment was conducted to establish a rapid in vitro clonal propagation of seven strawberry genotypes and their field evaluation under Bangladesh condition. Runner tips of seven strawberry genotypes viz. AOG, JP-2, JP-3, Camarosa, Sweet charley, Giant Mountain and Festival were cultured in vitro for multiple shoot proliferation and root induction. Proliferation of runner tips was obtained on Murashige and Skoog (MS) basal medium containing three different concentrations (1.0, 1.5, 2.0 mg/l) of BA with 0.5 mg/l 6-ferfuryl amino purine (KIN) or gibberellic acid (GA3). The best shoot proliferation was obtained from cultures grown on medium supplemented with 1.5 mg/l BA with 0.5 mg/l KIN. Microcuttings were rooted on half strength MS medium with 0.5 to 1.5 mg/l indole butyric acid (IBA) or indole acetic acid (IAA). Maximum rooting (90 to 98%) with 9 to 12 roots/cultures was achieved at 1.0 mg/l IBA. The plantlets, thus developed were hardened and successfully stablished in soil. AOG was found to be the most responsive genotype followed by JP-2 and JP-3.


INTRODUCTION
Strawberry (Fragaria × ananassa Duch.) belongs to the Rosaceae family. It is a perennial, stoloniferous herb. Strawberries have traditionally been a popular delicious fruit for its flavour, taste, fresh use, freezing and processing. It contains relatively high quantities of ellagic acid having a range of biological activity and especially the fruit contains higher vitamin C concentration than orange or lemon. It is produced in 73 countries worldwide on 200,000 hectors and produced 31 lac metric tons strawberry (FAO, 2008). It has been commercially cultivated in Canada, USA, Japan, Spain, Germany, Korea, Italy, Poland, Thailand and so many countries in the world .
Strawberry is traditionally propagated vegetatively by rooted runners but this method is not proved suitable due to incidence of many diseases infection and environmental hazards and resulting in the gradual degeneration of cultivers performance. Karhu and Hakala (2002) observed that micropropagated strawberry plants were comparatively better in different characters (crown size, number of runners, flowering time and yield of berries) than conventionally propagated runner plants.
Conventionally, strawberry is propagated by runners (Sakila et al., 2007), which is very labour intensive; time consuming and results in the transmission of viral diseases (Gautam et al., 2001).
In contrast of these, mass multiplication in vitro through tissue culture results high yield in disease free plant material (Mohan et al., 2005) and proved to be the best alternative approach to conventional propagation method (Mahajan et al., 2001). The standardization of protocol and procedure of micropropagation of strawberry was successfully attempted by many (Kaur et al., 2005;Sakila et al., 2007;Gantait et al., 2010). But complete field performance of micropropagated plants was not studied enough where extensive field evaluation is necessary for commercial utilization of tissue culture (Smith and Hamill, 1996). Moreover, the conventional way of production is not adequate to meet the commercial demand. Micropropagated strawberry plant has been introduced to prevent most of the plant and soil transmissible diseases (Biswas et al., 2008).
For better strawberry production photoperiod 10 to 20 h, day temperature 12 to 30°C and number of short days 12 to 24 are essential (Michel et al., 2006). Bangladesh is a sub tropical country and here in winter average day temperature is 15 to 25°C, photoperiod 12 to 16 h and short days about 30 to 50 days (Biswas et al., 2008). Therefore in winter season, strawberry can be grown and nowaday it becomes very popular due to attractiveness of fruits, fragrance and nutritional quality. Since last few years, strawberries are cultivated but the main constrain of its cultivation is to maintain plant materials due to hot summer in Bangladesh. Therefore, in the present investigation an attempt was made to develop an efficient method of in vitro plant regeneration of strawberry for mass production of planting materials for commercial cultivation in Bangladesh.

MATERIALS AND METHODS
Runnes with tips of seven strawberry varieties viz. AOG, JP-2, JP-3, Camarosa, Sweet charly, Giant Mountain and Festival were collected from strawberry germplasm stocks maintained in Akafuji Agrotechnologies, Rajshahi, Bangladesh. AOG, JP-2 and JP-3 are Japanese varieties and Camarosa, Sweet charly, Giant Mountain and Festival are American varieties. Fresh runner tips from 2 months old mature strawberry plants ( Figure 1A) were collected during the first week of November 2007. Runner tips of seven strawberry varieties were washed first under running tap water for 30 min and treated with 1% Tween 80 for ten minutes followed by repeated rinsing with sterile distilled water. Further sterilization was done under aseptic condition in laminar air flow cabinet. Explants were surface sterilized with 50% (v/v) ethyl alcohol (1 min) followed by 0.1% (w/v) HgCl2 (4 min).
Finally, the explants were washed thoroughly (five times) with sterilized distilled water and cut into appropriate size (1.5 cm) and cultured on MS basal medium supplemented with specific concentration of growth regulators viz. 6-benzyladenine (BA), 6ferfuryl amino purine (KIN) and gibberellic acid (GA3) adding 30 g/l sugar (market sugar) and 0.8% agar (British Drug House, England).
The pH of the medium was adjusted to 5.7 before autoclaving at 1.06 kg/cm 2 and 121°C for 20 min. The cultures were incubated in growth chamber 16/8 light/dark cycle at 25±2°C. Proliferated multiple shoots after elongation were cut and individual shoots were placed in half strength MS medium containing different concentration of indole butyric acid (IBA) or indole acetic acid (IAA) for root induction. All chemical compounds including macro and micro nutrients, organic acids and inorganic acids, sugar, agar, KOH, HgCl2, ethanol etc. used in the present study were the reagent grade products of either BDH, England or MERCK, India. The vitamins, amino acids (Glycin), growth regulators were mostly products of Sigma Chemical Company; USA and Phytotec (USA) and a small portion of thiamine was a product of British Drug House (BDH), England. Data on shooting and rooting efficiency were recorded after 5 weeks of culture initiation. For each treatment 10 to 12 explants were used and the experiments were repeated three times.
Three-week-old rooted shoots were taken out from the culture tubes, thoroughly washed in water to remove agar gel and then transferred to plastic pot containing garden soil and compost (3:1 v/v) and were kept under transparent plastic shed to control the moisture condition. After one week plants were taken out from the shed and successfully survived plants were transferred to the field. Data were recorded from randomly selected ten plants/variety on plant height, no. of leaves/plant, no. of stolon/plant, no. of nodes/stolon, canopy size (cm 2 ), no. of flowers/plant, no. of fruits/plant and fruit weight/plant (g) to compare the varietal performance of the seven strawberry genotypes. No. of fruits/plant was recorded after 80 days of plantation and other characters were recorded after 60 days of plantation.

RESULTS AND DISCUSSION
Runner tips of seven strawberry varieties were inoculated on MS medium fortified with different concentration of BA (0.1, 0.5 and 2.0 mg/l) with KIN (0.1 and 0.5 mg/l) or gibberellic acid (GA 3 ) (0.1 and 0.5 mg/l). Within 8 to 14 days of culture multiple shoots emerged directly from the explants. The explants cultured in medium with 1.5 mg/l BA either with KIN or GA 3 initiated shoots 2 to 4 days early than other treatments. The rate of shoot proliferation ranged from 45 to 80% and the highest rate of response for all genotypes was obtained at 1.5 mg/l BA + 0.5 mg/l KIN combination (Table 1, Figure 1G). AOG showed the maximum frequency (88%) of shoot formation followed by JP-2 (86%) and JP-3 (80%). The number of shoots per explant ranged from 8.00 to 9.00 in 1.0 mg/l BA + 0.5 mg/l KIN and 5.00 to 6.00 in 1.5 mg/l BA + 0.5 mg/l GA 3.
In other treatments, the number of roots/plant was low. When BA concentration was increased from 1.0 to 1.5 mg/l, the number of shoots/explant increased but further increase of BA number of shoots decreased. JP-3 produced maximum number of shoots/explant in 1.5 mg/l BA + 0.5 mg/l KIN followed by AOG and JP-2. Hu and Wang (1983) reported that high concentration of cytokinin reduced the number of micropropagated shoots. Similar results have already been reported in Fragaria indica Andr. (Bhatt and Dhar, 2000). Also, this result is in consistent with the findings in papaya (Conover and Litz, 1978) as well as in Eucalyptus grandis (Teixetra and Silva, 1990). The developing shoots were elongated by subculturing on the same combinations of growth regulators. Later on elongated shoots were excised and used for root induction. Bhatt and Dhar (2000) reported multiple shoot regeneration from Indian wild strawberry using MS supplemented with 4.0 mg/l BA and 0.1 mg/l napthalene acetic acid (NAA). Some workers also reported shoot regeneration in strawberry using MS medium containing BA in combination with KIN (Lee and de Fossard, 1977;Sobczykiewicz, 1980;Lis, 1990;Boxus, 1999;Neeru et al., 2000;Mereti et al., 2003). Our results indicated that, low concentration of BA alone or with KIN were found suitable for shoot initiation and further multiplication. This difference may be attributed by the difference of genotype and physiological condition of the explants. The daughter shoots (3 to 4 cm length) were excised and transferred to root induction media. Both IBA and IAA were found to be effective for adventitious root induction and frequency of root induction ranged from 78 to 95%. Out of two concentrations of IBA or IAA 1.0 mg/l was proved to be superior where the shoots produced roots early and between the two auxins IBA showed better performance for root induction than IAA ( Figure 1H). AOG produced highest number of roots per shoot with highest frequency (95%) of root induction in medium fortified with 1.0 mg/l IBA (Table 2). No difference was observed in root length in this experiment. Similar effects of IBA were also observed in Calotropis gigentea (Roy and De, 1986), Capsicum annum (Agarwal et al., 1989) and Prunus sp. (Mante et al., 1989).
Rooted plantlets ware taken out from culture tubes and washed thoroughly with tap water to remove the culture  medium from the roots. Washed plantlets were sprayed with fungicide and planted to normal and sterilized soil in thump pot ( Figure 1J). After 7 days, the hardened planlets were planted in soil. After two months of planting, different agronomic characters of seven genotypes of strawberry were noted. It was exhibited from the result that plant growth showed wide range of variation for different agronomic characters except plant height (

ACKNOWLEDGEMENT
The authors thankfully acknowledge the Ministry of